Identification of crystals in synovial fluid

Synovial fluids can contain a number of crystals and other particulate matter. Some of these, particularly monosodium urate
monohydrate (MSUM) and calcium pyrophosphate dihydrate (CPPD) crystals, are pathogenic; others, including cholesterol and
other lipid particles and the basic calcium phosphates (BCPs), including apatites, are of doubtful significance. Table 1 lists the main forms of particulate matter that have been identified in synovial fluids.

Intrinsic and extrinsic crystals and particles detected in synovial fluid

In the case of MSUM and CPPD crystals it is clear that the identification of these crystals in a synovial fluid that also
has a high polymorphonuclear cell count (indicative of acute inflammation) is the only certain way to diagnose an attack of
gout or pseudogout respectively.1 Furthermore, this is one of the few tests that has been shown to change clinical practice in rheumatology,2 and it is apparent that the consequences of getting the diagnosis wrong can be severe.3 Therefore, accurate identification of MSUM and CPPD is important.

How can crystals be identified?

There are a large number of techniques that can be used to identify crystals that can be found in synovial fluid, nearly all
of which rely on microscopy of one sort or another because of the small size of the individual particles. They range from
the very simple, like Garrod’s famous “string test”,4 to the furiously complex, such as laser microscopy or atomic force microscopy.56 In clinical practice we need a relatively simple, affordable technique, with a reasonable degree of sensitivity and specificity.
Polarised light microscopy remains the only possibility that comes anywhere near fulfilling these needs: it is available in
most hospitals and is relatively inexpensive; in addition, as MSUM and CPPD crystals can be distinguished through their different
polarisation properties, it should, in theory, be reasonably reliable for these two crystal types (figs 1 and 2). But polarised light microscopy is not a crystallographic technique (and therefore cannot permit certain characterisation),
and it is virtually useless for the identification of basic calcium phosphates (BCP). However, we have yet to be convinced
that the identification of apatite or other BCP particles in the synovial fluid ever should or does affect patient management,
which renders it irrelevant to clinical practice.

Monosodium urate monohydrate crystals from a gouty tophus viewed under polarised light. Note the strong birifringence, needle
shaped morphology and “flaring” that help distinguish them from other crystals.

Calcium pyrophosphate dihydrate crystals extracted from the synovial fluid of a patient with pseudogout viewed under polarised
light microscopy. Note the variation in size and morphology of the crystals, and the fact that some of the particles do not
appear to exhibit birifringence.

We therefore recommend that clinical rheumatologists should only use polarised light microscopy to identify MSUM and CPPD
crystals, and to differentiate them from “look-alikes”, while ignoring the issue of BCP crystals and all other techniques
for crystal identification.

Is routine polarised light microscopy reliable?

No.

In the past it has often been the case that writers of reviews and textbooks (other than our own) have indicated that the
identification of MSUM and CPPD by polarised light microscopy is simple and easy, when in fact it is not. The veneer of simplicity
may arise from the fact that it tends to be those with skill and experience who write the reviews, and because the visualisation
of a sample packed full of urate needles is both simple, seductive and immensely satisfying.

However, several quality control exercises have indicated that inter-laboratory reliability is very poor.7-9 When synovial fluid samples containing a variety of different crystals have been passed around different laboratories every
type of error has commonly occurred—false negatives, false positives and misclassification—clearly the technique lacks both
sensitivity and specificity. We have a problem.

Why is polarised light microscopy unreliable?

There are a number of reasons for the problem.

The lack of sensitivity is largely attributable to the fact that crystals may be either too small (individually) or too sparse
(low concentration) to be identified. There is evidence in support of each of these contentions. Gordon et al10 added varying concentrations of crystals to synovial fluids and found that microscopists had an identification threshold
of around 10–100 μg/ml, whereas experiments with extraction have shown that in vivo concentrations may be as low as 2–3 μg/ml.
In addition, we and others have found that CPPD crystals range in length from around 0.4 to 20 μm, and that the majority are
sometimes below the 1 μm threshold that would allow identification by light microscopy.1112 This explains false negatives. False positives probably arise from the misclassification of contaminants as MSUM or CPPD
crystals, and is therefore a matter of skill and experience.

The lack of specificity, leading to misclassification of particles, can also be explained by lack of experience and skill.
More problems arise with CPPD than with MSUM. CPPD crystals are only weakly birifringent, and are probably better identified
by non-polarised light and characterised by morphology than by their polarisation properties.13

What can and should we do about the problem?

We must get our house in order.

Recent headlines about other laboratory tests, such as cervical smears, indicate that the widespread use of a test that changes
patient management, but is being carried out with a lack of specificity and sensitivity will no longer be tolerated. Nor should
it. We must get our house in order.

The sensitivity problem cannot be solved without the routine use of very complex techniques such as crystal extraction from
synovial fluids14 and analytical electron microscopy15—and this is impractical. However, we can and should always remember that the presence of crystals in synovial fluids is not
an “on-off” phenomenon and that there is a threshold for identification. In practice, it is unlikely that an episode of gout
or psuedogout will generally be driven by crystals that are either too small or too low in concentration to be identified
during the episodes, but this will often be the case between acute episodes of synovitis. As rheumatologists we must recognise
that a negative report from the examination of a synovial fluid by polarised light microscopy, however experienced the operator,
does not exclude the presence of small numbers of MSUM or CPPD crystals, or of many very small crystals of these salts.

The specificity problem is one that we can do something about. This revolves around experience, technique, equipment and skills.
The service could, in our view, be greatly improved by making sure that laboratories that take on this work have a good microscope,
a specially trained and experienced person to carry out the microscopy, and that they carry out good practice in this field.
This is something that EULAR has helped to kick start and that we and others are hoping to continue.

The EULAR initiative

During and after the Amsterdam EULAR Meeting (1993), disquiet was voiced about the poor quality of routine polarised light
microscopy for the identification of MSUM and CPPD. As a result, a small grant was awarded to Elisio Pascual and ourselves
to kick start an initiative to develop training, guidelines and quality control in this field.16 The products to date include a published booklet,13 which will soon be available on the EULAR web site, the completion of a number of training days (including one at the Vienna
EULAR meeting in 1997), and similar courses are now being put on by Ralph Schumacher in the USA among others. We are now progressing
towards establishing a quality control laboratory in Bristol. We intend to use this to make samples available to collaborating
laboratories for quality control, as well as developing and implementing good practice guidelines, and carrying out training,
as occurs in many other laboratory practices. Contact us if you would like to be a part of this ongoing work.

Conclusions

The only crystals worth looking for in synovial fluids are MSUM and CPPD, as these are the only ones whose identification
can and should change clinical practice.

Polarised light microscopy remains the only practical way of identifying these particles in the clinical setting.

Polarised light microscopy is a test with major problems in both its sensitivity and specificity for the identification of
these crystals.

The sensitivity problem is in part attributable to the problems caused by low concentrations of crystals or the presence of
crystals of very small size, a negative report could always be a false negative therefore, and crystal presence cannot be
regarded as an “on-off” phenomenon.

The specificity problem can be improved by the use of good equipment, good laboratory practice, specific training and quality
control exercises—initiatives to develop these improvements need supporting.